Fatty acid regulation of adhesion molecule expression in human vascular cells.

Atherosclerosis is a chronic inflammatory disease of the large and medium sized elastic arteries and is the primary cause of death in the western world. It is the underlying cause of heart disease and stroke. Endothelial dysfunction is the initial step of atherosclerosis, which leads to the formation of lesions at vulnerable sites such as branches and curvatures. Endothelial dysfunction produces an endothelium with pro-coagulant properties, which releases inflammatory cytokines and vasoactive molecules that produce an inflammatory reaction. The endothelium becomes increasingly permeable to LDL and an increase in adhesion molecule expression leads to leukocytes being recruited from the blood flow into the arterial wall. If the inflammatory response continues unabated an atherosclerotic lesion is formed which can progress from its smallest form, a fatty streak to an advanced, complicated and calcified lesion. Adhesion molecules control the binding of the leukocytes to the endothelium and their subsequent extravasation in to the arterial intima. They are expressed on the activated endothelium and on leukocytes. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin are expressed on endothelial cells and lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4), are expressed on the leukocytes. The consumption of n-3 polyunsaturated fatty acids (PUFA) from fish and fish oil have been shown to play an important role in the amelioration and prevention of cardiovascular disease and a number of inflammatory disorders. Similarly, conjugated linoleic acids (CLAs) from dairy products and the meat of ruminant animals have recently been shown to have anti-cancer properties. Studies using animal models have shown that CLA may also be beneficial in inhibiting the progression and inducing the regression of cardiovascular disease. In this study, the effects of both n-3 PUFA and CLA were assessed in human umbilical vein endothelial cells (HUVEC), smooth muscle cells (SMC) and THP-1 monocytes. The expression of the adhesion molecules was assessed by fluorescence activated cell sorting (FACS) on cell treated with n-3 PUFA and CLA for 24 hours followed by 6 hours cytokine stimulation. ICAM 1, VCAM-1 and E-selectin was measured on HUVEC and LFA-1, VLA-4, ICAM-1, VCAM-1 and E-selectin were assessed on THP-1 cells. The expression of ICAM-1, VCAM-1 and E-selectin was strongly induced with TNF-alpha and IL-6 stimulation. n-3 PUFA treatment decreased ICAM-1, VCAM-1 and E-selectin surface protein expression on TNF-alpha and IL-6 stimulated HUVEC. CLA isomers decreased ICAM-1, VCAM-1 and E-selectin surface protein expression on TNF-alpha stimulated HUVEC. Interestingly, TNF-alpha, IL-1Beta and IL-6 failed to increase the expression of any adhesion molecules on THP-1 monocytes. Furthermore, n-3 PUFA and CLA had no effect LFA-1 or VLA-4 expression on THP-1. This supports the theory that n-3 PUFA are beneficial in preventing the recruitment of leukocytes from the blood to within the arterial wall. Additionally, the results highlight the possibility that CLA may have similar beneficial properties to n-3 PUFA in relation to cardiovascular disease and support the theory that other classes of fatty acid apart from n-3 PUFA may exert influence on inflammation thereby suggesting the possibility of a common mechanism of action. Adhesion molecule expression in cells is controlled by various transcription factors, including nuclear factor kappa B (NF-kB), activating protein-1 (AP-1) and peroxisome proliferator-activated receptor (PPAR). To test the theory that n-3 PUFA and CLA are exerting their effects on adhesion molecule expression at a transcriptional level in HUVEC, a specific ELISA was used to test the DNA binding activity of NF-kB, AP-1 and PPAR. Cells were treated with n-3 PUFA and CLA for 24 hours followed by 6 hours TNF-alpha stimulation. Stimulation of HUVEC with TNF-alpha increased the active binding of the NF-kB subunits p50 and p65. Treatment with the n-3 PUFA eicosapentaenoic acid (EPA) significantly reduced p50 DNA binding activity and docosahexaenoic acid (DHA) and alpha-linolenic acid (LNA) significantly reduced NF-kB p65 binding activity. This suggests that the NF-kB dimer of p50/p65 is split on activation or homodimers are activated/inhibited. Interestingly, CLA had no significant effect on NF-kB p50 or p65 DNA binding activity. Stimulation of HUVEC with TNF-alpha increased the activity of the AP-1 subunits FosB, cJun, JunB and JunD. Treatment with n-3 PUFA followed by TNF-alpha stimulation decreased AP-1 subunit DNA binding activity to a greater extent than CLA treatment followed by TNF-alpha stimulation. Furthermore, the anti-atherosclerotic transcription factor, PPARy DNA binding activity, but not PPARalpha, was significantly increased by CLA, a known PPAR agonist. The results demonstrate that n-3 PUFA and CLA are able to alter the DNA binding activity of transcription factors to differing extents. Oxidative stress is another factor that is thought to contribute to the development and progression of atherosclerosis. The oxidative stress hypothesis for the development of atherosclerosis focuses on the oxidation of LDL within the arterial wall by reactive oxygen species (ROS). Cellular damage caused by increased ROS levels is limited by intrinsic antioxidant enzymes such as glutathione peroxidase (GPx1). It has previously been shown that n-3 PUFA and inflammatory cytokines are able to increase GPx1 mRNA levels in HUVEC. To study the impact of n-3 PUFA and CLA on GPx1 protein expression and activity the cells were treated for 24 hours with either n-3 PUFA or CLA. The cells were then stimulated for a further 6 hours with TNF-alpha. GPx1 protein expression was measured using a specific ELISA and GPx1 activity was analysed using an enzyme linked spectrophotometric assay. GPx1 protein expression was not altered by treatment with n-3 PUFA, CLA or TNF-alpha stimulation. However, GPx1 activity was significantly increased by treatment with EPA followed by TNF-alpha stimulation. CLA treatment had no effect on GPx1 activity. Unfortunately, it was identified that the protein and selenium levels of the samples were too low to determine any consistent effects that the fatty acid treatments and cytokine stimulation may have had on redox enzyme expression. However, Increased cell numbers resulted in an increase in GPx1 activity. This would result in increased reliability and reproducibility of the assays in the future. It is possible that n-3 PUFA and CLA may increase GPx1 protein expression and activity in HUVEC as has been shown with phospholipid hydroperoxide glutathione peroxidease (GPx4). The aims of this project were to achieve a greater understanding of the mechanisms by which n-3 PUFA and CLA exert their influence on vascular cell functions and atherosclerosis. The above in vitro results give an indication as to the cellular mechanisms that may occur in vivo. The results offer new avenues of investigation for the cellular mechanisms altered by the new groups of beneficial fatty acids obtained from the diet. These can be enhanced in dietary constituents and used as functional foods or nutraceuticals when consumed at higher concentrations than those found naturally in the diet.

Grantor dc:publisher.institution

Robert Gordon University

Year dc:date.issued

2007

Author dc:creator

• Mulgrew, Scott

Advisor dc:contributor.advisor

• K.W.J. Wahle, R. Knott and A. Sneddon

• Atherosclerosis
• Endothelial dysfunction
• Adhesion molecules
• n-3 polyunsaturated fatty acids (PUFA)
• Conjugated linoleic acid (CLA)
• Transcription factors
• Human vascular cells

Language dc:language

en

Identifier

oai:rgu-repository.worktribe.com:2807497
https://doi.org/10.48526/rgu-wt-2807497

OAI identifier oai:identifier

oai:rgu-repository.worktribe.com:2807497